Binding protein drug conjugates comprising anthracycline derivatives

ABSTRACT

The present invention relates to an anthracycline (PNU) derivative conjugate comprising a derivative of the anthracycline PNU-159682 having the formula (i) or formula (ii) which further comprises a linker structure X-L1-L2-L3-Y.

The present invention relates to binding protein drug conjugatescomprising anthracycline toxin derivatives

INTRODUCTION

Covalent conjugates of small molecular weight toxins (MW preferably<2,500 daltons) to binding proteins, in particular to antibodiesspecific for tumor cells, are powerful tools to specifically targetcancer cells for their destruction. Therefore, such binding protein drugconjugates (BPDCs), particularly antibody drug conjugates (ADCs), are ofhigh medical and commercial interest for the therapy of cancer. In orderto develop effective and safe BPDCs or ADCs for cancer therapy, severalaspects need to be addressed: First, the binding protein or antibodyneeds to be specific for a given tumor specific antigen (TSA), whichshould hardly or ideally not be expressed by normal or healthy tissuecells. Second, the covalent bond, or linkage, between the drug and thebinding protein needs to have the functionality to be stable enough incirculation, preventing undesired release of the toxic payload in theblood stream, but it has to effectively release the drug upon binding toand/or internalization into the cancer cells. Third, the toxic payloadhas to be of high enough toxicity, or potency, in order to effect thedestruction of the cancer cells, even if potentially limited amounts ofthe TSA are expressed on the cancer cells and therefore only limitedamounts of the ADC are internalized, or if release of the toxic payloadis not effected at high enough efficiency upon binding to the cancercells, or upon internalization into the cancer cell.

While the first aspect of successfully targeting cancer via a TSAdepends on a deep understanding of the target biology and the targetingmolecules developed for its specific binding, the second and thirdaspects, related to optimal linker and to toxin payload, generallyapplies to the effectiveness of binding protein drug conjugates (BPDCs)or antibody drug conjugates (ADCs).

All ADCs currently in clinical trials, and the two ADCs that areFDA-approved for cancer treatment, anti-CD30 ADC Adcetris®(brentuxumab-vedotin) from Takeda, and anti-HER-2 ADC Kadcyla®(trastuzumab emtansine, or T-DM1) from Roche/Genentech (see Perez et al.2014), are generated by chemical conjugation of toxic payloads involvingmaleimide linker chemistry either to primary amino groups of lysineresidues of the antibody, or to free thiol groups, generated by mildreduction of antibody intra-chain disulfide bridges. The chemicalconjugation has two limitations: First, it has been found that chemicalmaleimide-based linkers are associated with an undesired instability inthe presence of human serum albumin and thus lead to release of toxinsin circulation of patients treated with maleimide-linker containing ADCs(see Alley et al., 2008). Second, classical chemical conjugation bymaleimide linker chemistry results in heterogeneous BPDCs or ADCs,because it cannot be controlled to which amino- or thiol groups theconjugation occurs. Therefore, a Gaussian distribution of number ofdrugs covalently bound per antibody is obtained, such that conjugatedADCs have an average drug-to-antibody ratio (DAR) ranging between 3.5and 4. However, individual conjugates may have no drug attached (DAR=0)to the antibody, or up to 8 drugs attached to the antibody (DAR=8) incase of cysteine conjugates and even more drugs per antibody (>DAR 10)in case of lysine conjugates. Classical chemically conjugated ADCstherefore represent a heterogeneous mixture of different moleculesexhibiting different functional properties (see Panowski et al., 2014),which clearly is undesirable from a regulatory point of view indeveloping ADCs for treatment of cancer patients.

Therefore, there is a commercial and medical need to provide ADCs orBPDCs that are site-specifically conjugated, and thus are homogeneouswith regard to the drug-to-antibody ratio.

In addition, there is a commercial and medical need to provide ADCs orBPDCs with more stable drug to protein linkage that are more stable inblood circulation than the traditional conjugates based on maleimidelinker chemistry.

Further, there is a commercial and medical need to provide ADCs or BPDCsthat have a higher efficacy and less side effects than ADCs or BPDCscurrently on the market.

General Features of the Invention

The present invention solves these problems. It provides now toxins foruse in binding protein-drug conjugates, plus optionally a new technologyto conjugate these toxins to the said binding proteins in asite-specific manner by avoiding classical maleimide linker chemistry.

The general advantages of these two features will be discussed in thefollowing:

Linker Technology

Both, the above-mentioned serum instability and the heterogeneity ofchemically conjugated, and maleimide-linker containing BPDCs or ADCsrepresent significant liabilities for the safety of these drugs incancer patients, because both add to the non-specific release of thetoxin (“de-drugging”) of such ADCs in patients.

On one hand, the classical maleimide linkers can be broken up by freethiols in human serum, in particular cysteine-34 of human serum albumin,which—as the most abundant serum protein—provides the highestconcentration of free thiols in human serum. Cysteine-34 of human serumalbumin can break the thioether bond of maleimide linkers by way of aso-called retro-Michael reaction upon which the toxin is transferred andcovalently coupled to human serum albumin (HSA). The toxin-HSA conjugatecan then distribute the toxin in circulation or in the body without anytumor selectivity (see: Alley et al., 2008).

On the other hand, the higher DAR-species in the chemically conjugated,heterogeneous ADCs are known to have shorter serum half-lives due to ahigher hydrophobicity of these ADCs and a propensity for aggregation.Therefore these higher DAR species are subject to faster clearance fromserum, degradation and release of the toxin prior to the binding ofthese ADC to target positive cancer cells. In addition, higher DARspecies are also known to lead to a faster de-drugging, becauseindividual conjugation sites have different de-drugging kinetics,depending on the structural context of the amino acid carrying thetoxin.

The above-mentioned liabilities of chemically conjugated ADCs haveimpeded success of developing ADCs into the clinic, despite the factthat the concept of delivering a highly potent cellular toxin to cancercells via its coupling to a tumor cell-specific antibody is compelling.Due to toxin-related side-effects the first ADC that had beenFDA-approved in 2000, Mylotarg® (gemtuzumab ozogamicin) fromPfizer/Wyeth, needed to be taken off the market 10 years afterFDA-approval(http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm216448.htm).

The liabilities of chemically conjugated ADCs therefore restrictscurrent ADC development efforts to toxins with intermediate cellulartoxicity, like e.g. tubulin polymerization inhibitingdolastatin/auristatin-based and maytansin-based drugs. In fact, morethan 90% of all ADCs currently in clinical evaluation carry toxinsrelated to monomethyl auristatin E (MMAE) or F (MMAF) or to maytansine(e.g. DM1 or DM4) (see: Mullard (2013))

However, tubulin polymerization inhibiting drugs cannot reach potenciesbelow the nanomolar range, because tubulin, a component of the cellularcytoskeleton, is a highly abundant intracellular protein target, so thatmany drug molecules need to diffuse or be transported into the cell, inorder to shut down metabolism of the intracellular cytoskeleton,required for cell division and survival. The intermediate potency oftubulin polymerization inhibiting drugs, which can tolerate a certaindegree of “de-drugging” of conjugates, and their specific action ondividing and mitotic cells has made toxins with this particular mode ofaction most popular for the development of ADCs.

However, in order to specifically address tumors with low expressionlevel of TSAs, much higher potent toxins will be required. Therefore,newer ADC strategies, still in preclinical development involve toxinswith high cellular toxicity and different mode of actions, in particularDNA damaging toxins, like duocarmycins (see: Doktor et al. (2014) andpyrrolobenzodiazepines (PBDs) (see: Hartley & Hochhauser (2012)).

Anthracycline Derivatives

A highly interesting class of DNA intercalating toxins for use aspayloads for BPDCs or ADCs are anthracyclines, because of their provenclinical validation as chemotherapeutic drugs in cancer therapy (see:Minotti (2004)) Anthracyclines are red-colored polyketides with highanti-tumor activity, originally derived from Streptomyces species. Manyderivatives have been described during the last 40 years, including somethat are routinely used as chemotherapy drug for various solid andhematological cancers, e.g. doxorubicin (also called adriamycin),daunorubicin, epirubicin, idarubicin, or valrubicine. There is even oneanti-CD74 ADC in phase I/II clinical trials for multiple myeloma andother hematological cancers with doxorubicin as a toxic payload,milatuzmab-doxorubicin (see: clinicaltrials.gov identifier:NCT01101594).

All of the anthracycline-based chemotherapeutic drugs are known to showlimited potency on tumor cells as free drugs with IC₅₀s in the μmol/mlrange on most tumor cells (Crooke and Prestayko, 1981). Despite theexample of a first doxorubin-ADC currently evaluated in clinical trials,the use of conventional anthracyclines as toxic payloads for ADCstrategies is likely to remain challenging.

About a decade ago, a novel anthracycline derivative, called PNU-159682,has been described as a metabolite of nemorubicin (see: Quintieri et al.(2005) Clin. Cancer Res. 11, 1608-1617), which has recently beenreported to exhibit extremely high potency for in vitro cell killing inthe pico- to femtomolar range with one ovarian (A2780) and one breastcancer (MCF7) cell line (WO2012/073217 A1, Caruso et al.). The structureof the anthracycline derivative PNU-159682, as disclosed in theabove-mentioned prior art documents is disclosed in FIG. 2 for thepurpose of reference, and with the official anthracycline numberingsystem for reactive carbons of the tetracyclic aglycone structure.

Based on the above-mentioned limitations of chemically conjugated ADCs,with regard to maleimide-linker instability and de-drugging of higherDAR species in heterogeneous chemically conjugated ADCs, a highly potentanthracycline toxin, like PNU-159682, is expected to be highlyproblematic in the context of classical chemical conjugation, due torelease of the toxin in circulation prior to targeting of the tumorcells.

Therefore potent toxins, as e.g. PNU-159682, homogeneous ADCs withdefined pharmacokinetic properties and extended serum stability arerequired, in order to avoid, or to minimize side effects fromprematurely released toxins in circulation of patients. However, at thesame time, specific killing of tumor cells characterized by low targetexpression still needs to be possible.

Although the use of PNU-159682 as a payload for ADC generated byclassical chemical maleimide linker approaches has been disclosed before(WO2009/099741 A1, Cohen et al.), no functional data were provided inthis prior art document. First functional data with PNU-159682 linked toantibodies with different linker and spacer structures in the context ofchemically conjugated and heterogenous maleimide-linker containingconjugates on tumor cells were disclosed in the prior art documentWO2010/009124 A2 (Beria et al.), but safety and pharmacokinetic data wasnot provided.

PREFERRED EMBODIMENTS

According to a first preferred embodiment, anthracycline (PNU)derivative conjugates are described that contain PNU-159682 derivativeslacking the C14 carbon and attached hydroxyl group of the tetracyclicaglycone structure characteristic for anthracyclines. As a secondpreferred embodiment anthracycline (PNU) derivative conjugates aredescribed lacking both the C13 and C14 carbons with carbonyl function atC13 and hydroxyl group at C14 of the teracyclic aglycone structurecharacteristic for anthracyclines.

In these embodiments, the anthracycline (PNU) derivative conjugatescomprise a derivative of the anthracycline PNU-159682 having thefollowing formula (i) or formula (ii):

Said conjugates comprise at their wavy line a linker structure that canhave different elements, X-L₁-L₂-L₃-Y, wherein L₁-L₃ represent linkers,and two of L₁-L₃ are optional, and wherein X and Y further representeach one or more optional linkers.

Both derivatives are markedly different to PNU-159682, which is ametabolite of the anthracycline nemorubicin and has for the first timebeen disclosed by Quintieri et al. (2005).

Both C13 and C14 carbons with their carbonyl function at C13 and thehydroxyl group at C14 are a mandatory structural feature of PNU-159682,which are not part of the derivative conjugates disclosed herein.

Surprisingly, and for the first time, it is demonstrated thatPNU-derivatives without carbon 14 and attached hydroxyl group of thetetracyclic aglycone structure characteristic for anthracyclines exhibitcellular toxicity, e.g., in site-specifically conjugated antibody drugconjugates. Preferred embodiments thereof are shown in FIGS. 3A, 6A and6B.

According to another embodiment of the invention, a binding protein-drugconjugate (BPDC) is provided, having the following formula:

-   -   wherein L₁-L₃ represent linkers, and two of L₁-L₃ are mandatory,    -   wherein X any Y represent each one or more optional linker,    -   wherein BP is a binding protein, and    -   wherein n is an integer between ≧1 and ≦10.

In this construct, several linkers can form a unitary chain thatconjugates one toxin to the one binding protein, and/or several linkerscan connect several toxins to the one binding protein. Likewise, thelinkers can conjugate two or more subunits of the same binding proteinto two or more toxin molecules.

The optional linker X can be any chemical linker structure known in theprior art, that have been used in ADCs to allow specific release of thetoxin upon internalization into cancer cells (see e.g. Ducry & Stump(2010) or McCombs et al. (2015)

Some examples for such linkers described in the prior art, which areonly provided by way of example and not intended to be limiting, areshown below.

Linkers L₁, L₂ and L₃ are discussed below.

The optional linker Y can be any chain of amino acids with up to 20amino acids allowing optimal conjugation of the binding protein to theunitary chain of linkers X, L₁, L₂, L₃ or variations thereof, inparticular to L₃.

Furthermore, linker structures are provided, that allow site-specificconjugation of the PNU-derivatives to suitable binding proteins, e.g,and preferably to antibodies. The derivatives can thus be used toproduce site-specifically conjugated, homogeneous binding protein-drugconjugates, which can be used in therapeutic applications, like anticancer therapy.

According to another preferred embodiment of the anthracycline (PNU)derivative, the linker structure comprises, as L₂, an oligo-glycinepeptide (Gly_(n)) coupled to said anthracycline derivative, directly orby means of another linker L₁, in such a way that the oligo-glycine(Gly_(n)) peptide has a free amino terminus, and wherein n is an integerbetween ≧1 and ≦21.

In each case (Gly)_(n) (also called (Gly)_(n)-NH₂ or Gly_(n)-stretchherein) is a an oligo-glycine peptide-stretch. In one particularlypreferred embodiment, n is an integer between ≧3 and ≦10, preferably ≧3and ≦6. Most preferred, n=5.

As already disclosed herein, the anthracycline (PNU) derivativesdisclosed herein are derivatives of PNU-159682 either lacking carbonatom 13 and 14 or lacking only carbon 14 with attached functionalgroups.

With respect to formula (i), it is one preferred embodiment that theoligo-glycine peptide (Gly)_(n) (≧1 and ≦21, preferably n=3 or n=5) isconjugated to the anthracycline derivative by means of analkylenediamino linker (NH₂—(CH₂)_(m)—NH₂, m≧1 and ≦11, preferably m=2),which is conjugated to the anthracycline derivative by means of a firstamide bond to carbon 13 and conjugated to the carboxyterminus of theoligo-glycine peptide by means of a second amide bond. The preferredcompound, PNU-EDA-Gly₅, useful for generating site-specificallyconjugated anthracycline (PNU) derivative conjugates is depicted in FIG.3A.

With respect to formula (ii), it is a preferred embodiment that theoligo-glycine peptide (Gly)_(n) (≧1 and ≦21, preferably n=3 or n=5) isdirectly coupled to Ring A of the PNU derivative (or carbon 9 of theanthracycline aglycone structure), such that the carbonyl group ofcarbon 13 represents the carboxy-end of the glycine peptide linker. Thepreferred compound, PNU-Gly₅, useful for generating site-specificallyconjugated anthracycline (PNU) derivative conjugates is depicted in FIG.6A.

With respect to formula (ii), it is another preferred embodiment thatthe oligo-glycine peptide (Gly)_(n) (≧1 and ≦21, preferably n=3 or n=5)is conjugated directly to Ring A of the PNU derivative (or carbon 9 ofthe anthracycline aglycone structure), with a alkyleneamine linker—(CH₂)_(m)—NH₂, m≧1 and ≦11, preferably m=2) that is conjugated to thecarboxyterminus of the oligo-glycine peptide by means of an amide bond.The preferred compound, PNU-EA-Gly_(n) useful for generatingsite-specifically conjugated PNU-derivative conjugates is depicted inFIG. 6B.

In the following, the anthracycline derivative conjugates according tothe above description are also called “PNU-EDA-Gly_(n)-NH₂”,“PNU-Gly_(n)-NH₂” or “PNU-EA-Gly_(n)-NH₂”, or in short also“PNU-EDA-Gly_(n)”, “PNU-Gly_(n)”, or “PNU-EA-Gly_(n)”, respectively, orin its preferred embodiment with 5 glycine residues, “PNU-EDA-Gly5”,“PNU-Gly5”, or “PNU-EA-Gly5”, respectively.

The invention further provides a binding protein-drug conjugate (BPDC),comprising an anthracycline derivative conjugate according to the abovedisclosure, which derivative further comprises a binding proteinconjugated to the free amino terminus of the oligo-glycine peptide(Gly_(n)) by means of an additional amide bond.

According to another embodiment of the anthracycline (PNU) derivative orthe binding protein-drug conjugate (BPDC), the oligo-glycine peptide(Gly_(n)), designated as L₂, is conjugated to the anthracyclinederivative of formula (i) by means of an alkylenediamino linker,designated as L₁, which alkylenediamino linker is conjugated to theanthracycline derivative by means of a first amide bond, while it isconjugated to the carboxy terminus of the oligo-glycine peptide by meansof a second amide bond, said conjugate of alkylenediamino linker andoligo-glycine peptide having the following formula (v),

wherein the wavy line indicates the linkage to the anthracyclinederivative of formula (i).

m is an integer between ≧1 and ≦11, and n is an integer between ≧1 and≦21. Preferably, m is an integer between ≧2 and ≦4, most preferably m=2(ethylenediamino group, EDA).

The alkylenediamino linker is used to allow attachment of the (Gly)_(n)linker for sortase conjugation, such that the coupling can occur via theC-terminus of the (Gly)_(n) peptide, thus providing a free N-terminus ofthe final toxin-linker adduct for sortase conjugation. It is to beunderstood that any CH₂ methylene group in the alkylenediamino linkermay be substituted by another stable bond, e.g. an —O— (ether), —S—(thioether), —NH— (amine), or any other alkyl, hetero-alkyl, aryl orhetero-aryl group, or any combination thereof, in order to realize theinvention.

According to another embodiment of the anthracycline (PNU) derivative orthe binding protein-drug conjugate (BPDC), the oligo-glycine peptide(Gly_(n)) is directly coupled to Ring A (or carbon 9) of theanthracycline derivative of formula (ii). See FIG. 6A for anillustration thereof.

According to another embodiment of the anthracycline (PNU) derivative orthe binding protein-drug conjugate (BPDC), the oligo-glycine peptide(Gly_(n)) is conjugated to the anthracycline derivative of formula (ii)by means of an alkyleneamino linker, designated as L₁, whichalkyleneamino linker is conjugated to the carboxy terminus of theoligo-glycine peptide by means of an amide bond, said conjugate ofalkyleneamino linker and oligo-glycine peptide having the followingformula (vi)

wherein the wavy line indicates the linkage to the anthracyclinederivative of formula (ii), wherein m is an integer between ≧1 and ≦11,and n is an integer between ≧1 and ≦11. Preferably, m is an integerbetween ≧2 and ≦4, most preferably m=2 (ethyleneamino group, EA).

The alkyleneamino linker is used to allow attachment of the (Gly)_(n)linker for sortase conjugation, such that the coupling can occur via theC-terminus of the (Gly)_(n) peptide, thus providing a free N-terminus ofthe final toxin-linker adduct for sortase conjugation. It is to beunderstood that any CH₂ methylene group in the alkyleneamino linker maybe substituted by another stable bond, e.g. an —O— (ether), —S—(thioether), —NH— (amine), or any other alkyl, hetero-alkyl, aryl orhetero-aryl group, or any combination thereof, in order to realize theinvention.

In another embodiment of the binding protein-drug conjugate (BPDC), thelinker structure L₃ comprises a peptide motif that results from specificcleavage of a sortase enzyme recognition motif.

As disclosed elsewhere herein as well as in WO2014140317, content ofwhich is incorporated by reference herein, sortases (also called sortasetranspeptidases) form a group of prokaryotic enzymes that modify surfaceproteins by recognizing and cleaving a specific sorting signalcomprising a particular peptide motif. This peptide motif is also called“sortase enzyme recognition motif”, “sortase tag” or “sortaserecognition tag” herein. Usually, a given sortase enzyme has one or moresortase enzyme recognition motifs that are recognized. Sortase enzymescan be naturally occurring, or may have undergone genetic engineering(Doerr et al., 2014).

In a preferred embodiment of the binding protein-drug conjugate (BPDC),said said sortase enzyme recognition motif comprises a pentapeptide.

In preferred embodiment of the binding protein-drug conjugate (BPDC),said said sortase enzyme recognition motif comprises at least one of thefollowing amino acid sequences (shown N-terminus->C-terminus):

-   -   LPXTG    -   LPXSG, and/or    -   LAXTG.

The first two sortase enzyme recognition motifs are recognized by wildtype Staphylococcus aureus sortase A. The second one is also recognizedby engineered sortase A 4S9 from Staphylococcus aureus, and the thirdone is recognized by engineered sortase A 2A-9 from Staphylococcusaureus (Doerr et al, 2014). In all three cases, X can be any of the 20peptidogenic amino acids.

These sortase enzyme recognition motifs are, for example, fused to theC-terminus of a binding protein, or a domain or subunit thereof, bygenetic fusion, and are co-expressed therewith. Said fusion can be donedirectly, or indirectly, via additional linker Y described elsewhereherein,

It is noteworthy that, once integrated in the linker structure andconjugated to L₂, L₃ lacks the 5^(th) amino acid residue (C-terminal G)of the sortase enzyme recognition motifs. In table 1, said C-terminal Gis thus shown in parentheses. In case the sortase enzyme recognitionmotif is a pentapeptide, L₃ is thus a tetrapeptide.

Prior to sortase conjugation, the sortase enzyme recognition motifs mayfurthermore carry other tags, like His-tags, Myc-tags or Strep-tags (seeFIG. 4a of WO2014140317, the content of which is incorporated byreference herein), fused C-terminal to the sortase enzyme recognitionmotifs. However, because the peptide bond between the 4^(th) and 5^(th)amino acid of the sortase enzyme recognition motif is cleaved uponsortase mediated conjugation, these additional tags will eventually beremoved from the fully conjugated BPDC.

The sortase enzyme recognition motifs can be conjugated to the (Gly)_(n)linker that is conjugated to the anthracycline derivative by means ofthe sortase technology disclosed herein and in WO2014140317. During theconjugation process, one glycine reside from the (Gly)_(n) linker isreleased.

It is noteworthy to mention that, while these three peptide stretchesare shown above in the classical N-terminus->C-terminus direction, thatthe L residue is the one that is fused to the C-terminus of the bindingprotein, or to the C-terminus of linker Y, by means of a peptide bond.The 5^(th) amino acid residue (G) of L₃ is removed upon conjugation tothe (Gly)_(n) peptide, while the 4^(th) T or S amino acid residue of L₃is the one that is actually conjugated to the N-terminus of the(Gly)_(n) peptide.

The following table thus gives an overview of the preferred embodimentsof the Binding protein-drug conjugate (BPDC) of the invention, withL₁-L₃ shown.

TABLE 1 Typical linker structures L₃ (shown Binding Toxin L₁ L₂ hereC′−> N′) protein formula (i) alkylene- (Gly)n (G)TXPL antibody dieaminogroup (G)SXPL (G)TXAL formula (ii) alkyleneamino (Gly)n (G)TXPL antibodygroup (G)SXPL (G)TXAL

As discussed it is noteworthy that, once integrated in the linkerstructure and conjugated to L₂, L₃ lacks the 5^(th) amino acid residue(C-terminal G). In table 1, said C-terminal G is thus shown inparentheses.

According to another embodiment of the binding protein-drug conjugate(BPDC), the anthracycline (PNU) derivative is conjugated, by means ofthe one or more linkers, to the carboxy terminus of the binding protein,or to the carboxy terminus of a domain or subunit thereof.

In another preferred embodiment, n in the oligo-glycine (Gly_(n))peptide linker is an integer between ≧3 and ≦11, more preferably between≧3 and ≦7, preferrably n=3, or n=5. Most preferably, n in theoligo-glycine (Gly_(n)) peptide linker is 5.

In one preferred embodiment, the payload is the one of formula (i).

In a second preferred embodiment, the payload is the one of formula(ii).

According to another embodiment of the binding protein-drug conjugate(BPDC), the binding protein is conjugated to the free amino terminus ofthe oligo-glycine peptide (Gly_(n)) by means of an amide bond.

According to another embodiment of the binding protein-drug conjugate(BPDC), the binding protein is at least one selected from the groupconsisting of an

-   -   antibody,    -   modified antibody format,    -   antibody derivative or fragment retaining target binding        properties    -   antibody-based binding protein,    -   oligopeptide binder and/or    -   an antibody mimetic.

The term “binding protein”, as used herein, is equivalent to the term“immunoligand” as used in other publications by the inventors, includingthe appendix 1, which provides further technical details, disclosure andenablement as regards the sortase enzyme conjugation technology.

“Antibodies”, also synonymously called “immunoglobulins” (Ig), aregenerally comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains, and are therefore multimeric proteins, or anequivalent Ig homologue thereof (e.g., a camelid nanobody, whichcomprises only a heavy chain, single domain antibodies (dAbs) which canbe either be derived from a heavy or light chain); including full lengthfunctional mutants, variants, or derivatives thereof (including, but notlimited to, murine, chimeric, humanized and fully human antibodies,which retain the essential epitope binding features of an Ig molecule,and including dual specific, bispecific, multispecific, and dualvariable domain immunoglobulins; Immunoglobulin molecules can be of anyclass (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2) and allotype.

Provided the binding protein is an antibody, the bind protein drugconjugate is an antibody drug conjugate (ADC).

In the following, ADCs according to this invention are also called“PNU-EDA-Gly_(n)-Ab”, “PNU-Gly_(n)-Ab” or “PNU-EA-Gly_(n)-Ab”.

An “antibody-based binding protein”, as used herein, may represent anyprotein that contains at least one antibody-derived V_(H), V_(L), orC_(H) immunoglobulin domain in the context of other non-immunoglobulin,or non-antibody derived components. Such antibody-based proteinsinclude, but are not limited to (i) F_(c)-fusion proteins of bindingproteins, including receptors or receptor components with all or partsof the immunoglobulin C_(H) domains, (ii) binding proteins, in whichV_(H) and or V_(L) domains are coupled to alternative molecularscaffolds, or (iii) molecules, in which immunoglobulin V_(H), and/orV_(L), and/or C_(H) domains are combined and/or assembled in a fashionnot normally found in naturally occurring antibodies or antibodyfragments.

An “antibody derivative or fragment”, as used herein, relates to amolecule comprising at least one polypeptide chain derived from anantibody that is not full length, including, but not limited to (i) aFab fragment, which is a monovalent fragment consisting of the variablelight (V_(L)), variable heavy (V_(H)), constant light (CL) and constantheavy 1 (C_(H)1) domains; (ii) a F(ab′)2 fragment, which is a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a heavy chain portion of a F_(ab) (F_(d))fragment, which consists of the V_(H) and C_(H)1 domains; (iv) avariable fragment (F_(v)) fragment, which consists of the V_(L) andV_(H) domains of a single arm of an antibody, (v) a domain antibody(dAb) fragment, which comprises a single variable domain; (vi) anisolated complementarity determining region (CDR); (vii) a single chainF_(v) Fragment (scF_(v)); (viii) a diabody, which is a bivalent,bispecific antibody in which V_(H) and V_(L) domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with the complementarity domains of another chainand creating two antigen binding sites; and (ix) a linear antibody,which comprises a pair of tandem F_(v) segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementarity lightchain polypeptides, form a pair of antigen binding regions; and (x)other non-full length portions of immunoglobulin heavy and/or lightchains, or mutants, variants, or derivatives thereof, alone or in anycombination. In any case, said derivative or fragment retains targetbinding properties

The term “modified antibody format”, as used herein, encompassesantibody-drug-conjugates, Polyalkylene oxide-modified scFv, Monobodies,Diabodies, Camelid Antibodies, Domain Antibodies, bi- or trispecificantibodies, IgA, or two IgG structures joined by a J chain and asecretory component, shark antibodies, new world primateframework+non-new world primate CDR, IgG4 antibodies with hinge regionremoved, IgG with two additional binding sites engineered into the CH3domains, antibodies with altered Fc region to enhance affinity for Fcgamma receptors, dimerised constructs comprising CH3+VL+VH, and thelike.

The term “antibody mimetic”, as used herein, refers to proteins notbelonging to the immunoglobulin family, and even non-proteins such asaptamers, or synthetic polymers. Some types have an antibody-likebeta-sheet structure. Potential advantages of “antibody mimetics” or“alternative scaffolds” over antibodies are better solubility, highertissue penetration, higher stability towards heat and enzymes, andcomparatively low production costs.

Some antibody mimetics can be provided in large libraries, which offerspecific binding candidates against every conceivable target. Just likewith antibodies, target specific antibody mimetics can be developed byuse of High Throughput Screening (HTS) technologies as well as withestablished display technologies, just like phage display, bacterialdisplay, yeast or mammalian display. Currently developed antibodymimetics encompass, for example, ankyrin repeat proteins (calledDARPins), C-type lectins, A-domain proteins of S. aureus, transferrins,lipocalins, 10th type III domains of fibronectin, Kunitz domain proteaseinhibitors, ubiquitin derived binders (called affilins), gammacrystallin derived binders, cysteine knots or knottins, thioredoxin Ascaffold based binders, SH-3 domains, stradobodies, “A domains” ofmembrane receptors stabilised by disulfide bonds and Ca2+, CTLA4-basedcompounds, Fyn SH3, and aptamers (peptide molecules that bind to aspecific target molecules).

The term “oligopeptide binder”, as used herein, relates to oligopeptidesthat have the capacity to bind, with high affinity, to a given target.The term “oligo” refers to peptides that have between 5 and 50 aminoacid residues.

According to another embodiment of the binding protein-drug conjugate(BPDC), the binding protein binds at least one entity selected from thegroup consisting of

-   -   a receptor    -   an antigen    -   a growth factor,    -   a cytokine, and/or    -   a hormone.

This list defines the different types of targets the binding protein canbind to. As used herein, the term “receptor” means a cell surfacemolecule, preferably a cell surface molecule that (i) binds specific, orgroups of specific, signalling molecules (i.e. a receptor, like, e.g.,the VEGF receptor), and/or (ii) has no known ligand (i.e. an orphanreceptor, like, e.g. HER2/neu). The natural receptors are expressed onthe surface of a population of cells, or they merely represent theextracellular domain of such a molecule (whether such a form existsnaturally or not), or a soluble molecule performing natural bindingfunction in the plasma, or within a cell or organ. Preferably, suchreceptor is a member of a signalling cascade that is involved in aparticular pathogenic process (e.g., a receptor that belongs to asignalling cascade of a growth factor), or is expressed on the surfaceof a cell or particle that is involved in a pathological process, e.g.,a cancer cell.

As used herein, the term “antigen” means a substance that has theability to induce a specific immune response, and may include surfaceproteins or protein complexes (e.g. ion channels). Often times, antigensare associated to pathogenic entities, e.g., a cancer cell.

As used herein, the term “cytokine” refers to small cell-signalingprotein molecules that are secreted by numerous cells and are a categoryof signaling molecules used extensively in intercellular communication.Cytokines can be classified as proteins, peptides, or glycoproteins; theterm “cytokine” encompasses a large and diverse family of regulatorsproduced throughout the body by cells of diverse embryological origin.

As used herein, the term “growth factor” relates to naturally occurringsubstances capable of stimulating cellular growth, proliferation andcellular differentiation. Usually a growth factor is a protein or asteroid hormone. Growth factors are important for regulating a varietyof cellular processes.

As used herein, the term “hormone” relates to a chemical released by acell, a gland, or an organ in one part of the body that sends outmessages that affect cells in other parts of the organism. The termencompasses peptide hormones, lipid and phospholipid-derived hormonesincluding steroid hormones, and monoamines.

In case the binding protein binds a receptor or an antigen, the bindingprotein-drug conjugate (BPDC) can for example be directed to a specificsite, e.g., to a pathogenic entity, e.g., a cancer cell, where thepayload, e.g. a toxin is delivered. Thus, the systemic toxicity of thetoxin or the chemotherapeutic agent is reduced, while the localconcentration of the latter at the site of action is increased, thusproviding a better efficacy while side effects are reduced. Furthermore,a respective signalling cascade can be inhibited by the binding of thebinding protein. In case the payload is a marker the latter can thus beused to mark a specific site, e.g., a cancer cell characterized by agiven surface antigen detected by the binding protein, for diagnosis.

In case the binding protein binds a growth factor, a cytokine, and/or ahormone, the binding protein-drug conjugate (BPDC) can for example bedirected to the site the growth factor cytokine or hormone usually bindsto, in order to deliver the payload in a site-specific manner. Further,a respective signalling cascade can be inhibited by the binding of thebinding protein.

As used herein, the term “to bind” means the well-understood interactionor other nonrandom association between binding protein, e.g.,antibodies, or antibody fragments, and their targets. Preferably, suchbinding reaction is characterized by high specifity and/or sensitivityto the target. Preferably, the binding reaction is characterized by adissociation constant (Kd)≦10⁻³ M, preferably ≦10⁻⁴ M, ≦10⁻⁵ M, ≦10⁻⁶ M,≦10⁻⁷ M, ≦10⁻⁸ M, ≦10⁻⁹ M, and most preferred ≦10⁴⁸.

According to another embodiment, the binding protein has at least twosubunits.

In this embodiment, one subunit can be conjugated to a derivative of theanthracycline PNU-159682 disclosed herein (see FIGS. 3A and 6A and 6B).

Preferably, at least two different drugs can be conjugated to the atleast two subunits site-specifically. This option provides a versatiletoolbox with which a large variety of different binding protein-drugconstructs can be created.

Preferably, the at least two different drugs are drugs interfering withdifferent cellular pathways. This means that, next to the anthracyclinederivative conjugate disclosed herein, a second toxin can be conjugatedto another subunit of the same binding protein.

Such embodiment can be accomplished, e.g., by conjugating the twodifferent drugs to each the 2 light chains of a full-length antibody,and to the 2 heavy chains of a full length antibody, respectively, byutilizing two different sortase enzymes, recognizing different sortaserecognition motifs (“sortase tags”), plus an antibody that containsdifferent C-terminal modifications at heavy and light chains comprisingthe respective recognition motifs for said different sortase enzymes.

In such way, an Antibody Drug Conjugate can be created which is composedof each two full-length Ig light chains and Ig heavy chains, containingdifferent payloads covalently attached to said heavy and light chains.

Such embodiment results, preferably, in the site-specific conjugation ofthe at least two subunits for the generation of binding protein drugconjugates with site-specific and equal payload conjugation to each ofsaid subunits.

In one embodiment of the binding protein-drug conjugate (BPDC) thebinding protein binds HER-2. Preferably, the binding protein is anantibody specific for HER-2.

In this embodiment, the HER-2 specific antibody preferably

-   -   a) comprises the CDR regions 1-6 of trastuzumab (humanized        hu4D5)    -   b) comprises the heavy chain variable domain and the light chain        variable domain of trastuzumab    -   c) has an amino acid sequence identity of 90% or higher with the        regions or domains of a) or b)    -   d) is trastuzumab, or a target binding fragment or derivative        thereof, and/or    -   e) competes with trastuzumab for binding to Her-2

The anti-HER-2 monoclonal antibody trastuzumab binds to domain IV ofHER-2. Preferably, the anti-HER-2 antibody comprises the primary aminoacid sequences of IgH and IgL chains of FIG. 11 A (Seq ID Nos 1 and 2).The sequences of trastuzumab are also disclosed in drug bank accessionnumber DB00072 (BI0D00098, BTD00098), which is incorporated by referenceherein, as well as in the IMGT database (VH:http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637H& V_(L):http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637L).

In another embodiment of the binding protein-drug conjugate (BPDC) thebinding protein binds CD30. Preferably, the binding protein is anantibody specific for CD30.

In this embodiment, the antibody preferably

-   -   a) comprises the CDR regions 1-6 of brentuximab (chimeric cAc10)    -   b) comprises the heavy chain variable domain and the light chain        variable domain of brentuximab    -   c) has an amino acid sequence identity of 90% or higher with the        regions or domains of a) or b)    -   d) is brentuximab or a target binding fragment or derivative        thereof, and/or    -   e) competes with brentuximab for binding to CD30

The sequences of Brentuximab (clone cAc10), which is the antibodycomponent of the approved drug Adcetris/Brentuximab vedotin is disclosedin US2008213289A1.

Preferably, the anti-CD30 antibody comprises the primary amino acidsequences of IgH and IgL chains of FIG. 11 (Seq ID Nos 3 and 4).

Preferably, in these embodiments, the toxin is the one of formula (i),

-   -   L₁ is an ethylendiamino linker,    -   L₂ is an oligo-glycine (Gly_(n)) peptide linker (with n being        the preferred length of 5 amino acids), and    -   L₃ represents the amino acid residues 1-4 of a processed sortase        tag pentapeptide motif (i.e., devoid of the C-terminal G residue        (5^(th) amino acid residue), which removed upon sortase mediated        conjugation to the (Gly)n peptide,    -   Linker X is absent, and    -   Y is a 5 amino acid linker between the C-terminus of the Ig        light chain and L₃, having preferably the amino acid sequence        GGGGS.

Alternatively, the toxin is the one of formula (ii), while

-   -   L₁ is an ethylenamino linker,    -   L₂ is an oligo-glycine (Gly_(n)) peptide linker, (with n being        the preferred length of 5 amino acids),    -   L₃ represents the amino acid residues 1-4 of a processed sortase        tag pentapeptide motif (i.e., devoid of the C-terminal G residue        (5^(th) amino acid residue), which removed upon sortase mediated        conjugation to the (Gly)_(n) peptide,    -   Linker X is absent, and    -   Y is a 5 amino acid linker between the C-terminus of the Ig        light chain and L₃, having preferably the amino acid sequence        GGGGS.

The invention further provides a method of producing a bindingprotein-drug conjugate (BPDC) according to the above description,wherein a binding protein carrying a sortase enzyme recognition motif isconjugated, by means of a sortase enzyme, to at least one anthracyclinederivative conjugate which carries, as L₂, an oligo-glycine peptide(Gly_(n)).

The sortase technology, its advantages (site specific conjugation,stoichimetrically defined relationship between toxin and bindingprotein, high efficiency of conjugation) is in detail explained inapplication WO2014140317A1, the content of which is incorporated byreference herein. Further explanations with respect to the sortase tagsare found above,

It is a preferred embodiment of the present invention to conjugatePNU-derivative payloads by SMAC technology to the C-terminus of bindingproteins, and preferably to the C-terminus of antibody or immunoglobulinchains to at least one Ig light or Ig heavy chain. This is achieved bygenerating mammalian cell expression constructs for binding protein orimmunoglobulin subunits which encode for a C-terminal pentapetiderecognition motif for sortase enzymes directly following the C-terminusof the binding protein, or the polypeptide subunit of a multimericbinding protein, like e.g. an antibody.

It is to be understood that the pentapeptide motif of sortase A ofStaphylococcus aureus, which is LPXTG or LPXSG and which has beenmentioned before, is only provided as a non-limiting example and may bereplaced by any other pentapetide motif recognized by sortase enzymesfrom other species or other classes, like sortase B from Staphylococcusaureus, which recognizes the pentapeptide motif NPQTN. Also recognitionmotifs may be used that are recognized by engineered sortase enzymes,like e.g. LAETG, recognized by an engineered version of sortase A ofStaphylococcus aureus recently described by Dorr et al. (2014).

WO2014140317 further provides technical details, disclosure andenablement with regard to the sortase conjugation technology, which isalso called SMAC technology (sortase mediated antibody conjugationtechnology). This technology allows the conjugation of two entities, onemarked with a (Gly)_(n) stretch (as discussed for the toxin aboveherein) and one with a so-called sortase tag, which is a peptide tagthan can be attached, e.g., to a binding protein.

These sortase tags are oligopeptides, usually pentapeptide motifs, whichare fused to a first entity (here: the binding protein) that is to beconjugated to a second entity (here: the anthracyclin derivative), insuch way that the C-terminus of said sortase tags oligopeptides remainsfree. As disclosed in WO2014140317 this can be accomplished byexpressing the binding proteins from expression vectors encoding theadditional amino acids for the pentapetide sortase tag.

Such sortase tag is e.g, LPXTG or LPXSG (for sortase A fromStaphylococcus aureus), LPXSG (for engineered sortase A 4S9 fromStaphylococcus aureus described in Dorr et al., 2014), or LAXTG (forengineered sortase A 2A9 from Staphylococcus aureus described in Dorr etal., 2014) with X being any of the 20 naturally occurring amino acids.However, such sortase tags may differ in sequence for sortase enzymesfrom other bacterial species or for sortase classes, as disclosed inWO2014140317, and in the prior art (Spirig et al. 2011).

The second entity comprises a Glycine-stretch (Gly_(n)-stretch) with afree N-terminus (—NH₂), which Gly_(n)-stretch is an oligo-glycinepeptide. Preferably, n is an integer between ≧1 and ≦21. In oneparticularly preferred embodiment, n is an integer between ≧3 and ≦10,preferably n=3 or n=5. Most preferred, n=5.

The sortase enzyme is then capable of fusing the two entities to oneanother by means of a transpeptidation reaction, during which theC-terminal amino acid residue (e.g., the G in LPXTG) is cleaved of, andthen replaced by the first glycine of said glycine stretch.

In another preferred embodiment the pentapeptide recognition motif maydirectly be appended to the last naturally occurring C-terminal aminoacid of the immunoglobulin light chains or heavy chains, which in caseof the human immunoglobulin kappa light chain is the C-terminal cysteineresidue, which in case of the human immunoglobulin lambda light chain isthe C-terminal serine residue and which in the case of the humanimmunoglobulin IgG₁ heavy chain may be the C-terminal lysine residueencoded by human Fcγ1 cDNA. However, another preferred embodiment isalso to directly append the sortase pentapeptide motif to the secondlast C-terminal glycine residue encoded by human Fcγ1 cDNA, becauseusually terminal lysine residues of antibody heavy chains are clippedoff by prosttranslational modification in mammalian cells. Therefore, inmore than 90% of the cases naturally occurring human IgG1 lacks theC-terminal lysine residues of the IgG1 heavy chains.

In another preferred embodiment the pentapeptide recognition motif maybe appended to the C-terminus of a human immunoglobulin IgG₁ heavy chainwhere the C-terminal lysine residue encoded by human Fcγ1 cDNA isreplaced by an amino acid residue other than lysine.

We have described previously that in some cases (e.g. at the C-terminusof the Ig kappa light chains, (Beerli et al. 2015) it is beneficial toadd additional amino acids between the C-terminus of the binding proteinand the sortase tag, L₃. This has been shown to improve sortase enzymeconjugation efficiencies of payloads to the binding protein. In the caseof Ig kappa light chains, it was observed that by adding 5 amino acids(GGGGS) between the last C-terminal cysteine amino acid of the Ig kappalight chain and the sortase tag improved the kinetics of conjugation, sothat the C-termini of Ig kappa light chains and Ig heavy chains could beconjugated with similar kinetics (see: Beerli et al. (2015). Therefore,it is another preferred embodiment to optionally include a linker Y ofbetween ≧1 and ≦21 amino acids in between the last C-terminal amino acidof a binding protein or antibody subunit and the sortase tag, L₃.

The invention further provides the use of a binding protein drugconjugate (BPDC) according to the above description, or produced with amethod of the above description, for the treatment of a human or animalsubject

-   -   suffering from,    -   at risk of developing, and/or    -   being diagnosed for

a given pathologic condition.

The invention further provides the use of a binding protein drugconjugate according to the above description for the manufacture of amedicament for the treatment of a human or animal subject

-   -   suffering from,    -   at risk of developing, and/or    -   being diagnosed for

a given pathologic condition.

Preferably, the pathologic condition is a neoplastic disease. Morepreferably, the the neoplastic disease is

-   -   a cancer that has an HER-2 expression score of 1+, 2+ or 3+, as        determined by IHC or ISH, which cancer is preferably a breast        cancer    -   a cancer that is CD30 positive as determined by IHC, ELISA or        flow cytometry, preferably a lymphoma, more preferably a Hodgkin        lymphoma (HL) or a systemic anaplastic large cell lymphoma        (sALCL)

Determination of the HER-2 status can for example be determinedaccording to the ASCO/CAP guidelines, which are described in Wolff et al2013.

Determination of the CD30 status can for example be determined accordingto the method of Young 2014.

The invention further provides a pharmaceutical composition comprising abinding protein drug conjugate (BPDC) according to the abovedescription, or produced with a method of the above description, and atleast one other pharmaceutically acceptable ingredient.

FURTHER DESCRIPTION

In order to overcome the main limitations of traditional maleimidelinker chemistry for the generation of BPDCs and ADCs, we havepreviously developed an enzymatic approach for generating BPDCs or ADCsusing sequence-specific transpeptidase enzymes, either employing sortaseenzymes, or so-called split-inteins (see: WO2014140317A1). Inparticular, it could be demonstrated that site-specific conjugation ofsmall molecular payloads by sortase enzymes, in the context ofantibodies, referred to as SMAC-technology (sortase-mediated antibodyconjugation technology), results in ADCs that are equally potent aschemically conjugated ADCs in killing cancer cells in vitro, if the samebinding protein and the same payload is employed, Furthermore,SMAC-technology generated ADCs specific for the HER-2 target lead tosimilarly potent tumor regression in xenotransplantation models, if thesame targeting antibody (anti-HER-2 trastuzumab) and the same toxicpayload (DM1) was employed (WO2014140317A1). However, first, inSMAC-generated ADCs no maleimide linker chemistry was employed, andsecond, the conjugation reaction was performed in a site-specific mannerto the C-termini of either IgH or IgL chains of the antibody, so thatmore homogeneous ADCs have been obtained.

In case of SMAC-technology, the site-specific conjugation can beeffected by e.g. recombinant sortase A enzyme of Staphylococcus aureus,that specifically recognizes an LPXTG or LPXSG pentapeptide motif (X=anyof the 20 naturally occurring amino acids) and that can be appended to arecombinant antibody intended for conjugation. Sortase A then uses anoligo-glycine-stretch as a nucleophile to catalyze a transpeptidation,by which the amino group of the oligo-glycine effects a nucleophilicattack to the peptide bond between the threonine or serine and glycineof the LPXTG or LPXSG pentapeptide motif. This results in the breakageof that peptide bond and the formation of a new peptide bond between theN-terminal glycine of the oligo-glycine peptide (see FIG. 1), i.e.resulting in a transpeptidation.

While it has been shown that trastuzumab-DM1 conjugates generated bysortase-mediated conjugation have comparable potency to the chemicallyconjugated DM1 conjugates (T-DM1, or Kadcyla®, already applied in theclinic), higher potency of SMAC-technology generated ADCs has not beenachieved (WO2014140317A1). This would not have been expected, becausethe same targeting antibody and the same payload have been employed.

Based on this and also other experiments with different monoclonalantibodies specifically binding to other TSAs, that are potentiallyexpressed at lower levels on cancer cells than the HER-2 target, or thatare potentially less efficiently internalized upon ADC binding (data notshow), it became apparent, that toxic payloads with higher potency thanmaytansines, and/or with a potentially different mode of action arerequired to produce sufficiently effective ADCs. In addition, thepayload has to be amenable to modification at at least one reactivegroup, allowing the addition of an oligo-glycine peptide to enablesortase conjugation of the payload to LPXTG- or LPXSG-modified bindingproteins. Lastly, if higher potency toxins are employed, themodification should result in a stable linkage between theglycine-stretch and the payload, in order to prevent undesired releaseof the toxic payload in circulation, but at the same time the toxinshould still result in effective killing of cancer cells upon specificbinding and internalization of the BPDC or ADC into tumor cells.

Empiric evaluation of different toxic payloads described in the priorart in the context of SMAC-technology has resulted in the finding that ahighly potent anthracycline derivative of nemorubicin, called PNU-159682(Quintieri et al., 2005) (see also FIG. 2), that has been modified withan ethylenediamine-spacer, in order to allow addition of a pentaglycinestretch could very efficiently be conjugated to LPXTG modifiedantibodies by SMAC technology yielding almost completely conjugated ADCsbased on analyses of the products by HIC (hydrophobic interactionchromatography) and reverse-phase chromatography (data not shown). Inaddition, if this modified PNU-159682 derivative, termed PNU-EDA-Gly₅,is SMAC conjugated to various monoclonal antibodies, as described in theEXAMPLES, provided below, highly potent and TSA-dependent killing oftumor cells has been effected. In particular, HER-2 low expressing humanbreast cancer cells could efficiently be killed in vitro withSMAC-technology conjugated PNU-EDA-Gly₅, conjugates, whereasmaytansine-toxin conjugates were hardly effective. This demonstrates thepotential utility of the PNU-EDA-Gly₅ derivative for generating potentBPDCs and ADCs, preferably containing PNU-EDA-Gly₅, or anyPNU-derivative with a oligo-glycine peptide with at least two glycinesattached to it. In addition, it demonstrates the utility of BPDCs andADCs containing preferably PNU-EDA-Gly₅, or any PNU-derivative with aoligo-glycine peptide as the payload for the treatment of cancerdiseases.

Although anthracycline derivative PNU-159682 (FIG. 2) and its use in thecontext of chemical conjugation and ADCs have been described in theprior art (e.g. WO2009099741A1, WO2010009124, WO2012073217, provided forreference herein), a compound similar to PNU-EDA-Gly_(n), or ADCscontaining sortase-conjugated PNU-EDA-Gly_(n), as disclosed herein, havenot yet been described in the prior art, nor is the particular structureof the PNU-derivative with EDA spacer and Gly_(n) linker disclosed orclaimed in any of the prior art documents. Stable adducts, in which PNUderivatives are stably linked to proteins via peptide bonds rather thanby ester bonds and maleimide linkers may prove to be superior, in termsof stability and pharmacokinetic behavior in vivo, due to a generallyhigh stability of peptide bonds in serum, as disclosed in the Examplesfurther below. Additionally, PNU-derivatives with Gly_(n)-stretch thatare expected to display stable drug conjugates after SMAC-technologyconjugation are disclosed in FIG. 6A and FIG. 6B.

EXPERIMENTS AND FIGURES

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive; theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed invention, from a study ofthe drawings, the disclosure, and the appended claims.

In the claims, the word “comprising” does not exclude other elements orsteps, and the indefinite article “a” or “an” does not exclude aplurality. The mere fact that certain measures are recited in mutuallydifferent dependent claims does not indicate that a combination of thesemeasures cannot be used to advantage. Any reference signs in the claimsshould not be construed as limiting the scope.

All amino acid sequences disclosed herein are shown from N-terminus toC-terminus; all nucleic acid sequences disclosed herein are shown5′->3′.

Example 1: Generation of Site-Specifically C-TerminallyPNU-EDA-Gly_(n)-Payload Conjugated Monoclonal Antibodies Brentuximab andTrastuzumab by Sortase Mediated Antibody Conjugation Technology(SMAC-Technology)

The heavy and light chain variable region sequences of monoclonalantibody brentuximab (clone cAc10) specific for the human CD30 targetwere obtained from patent US2008213289A1, those of the human HER-2specific trastuzumab antibody contained in the commercial antibodyHerceptin (trastuzumab), or the ADC Kadcyla® derived thereof, werederived from the online IMGT database (V_(H):http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637H& V_(L):http://www.imgt.org/3Dstructure-DB/cgi/details.cgi?pdbcode=7637&Part=Chain&Chain=7637L.Chimeric mAb cAc10 and humanized mAb trastuzumab were produced withtheir heavy and light chains C-terminally tagged with a Sortase Arecognition sequence and an additional Strep II affinity purificationtag (HC tag sequence: LPETGGWSHPQFEK; LC tag sequence:GGGGSLPETGGWSHPQFEK) using methods known to those skilled in the art.(see FIGS. 11A & 11B).

The anthracycline derivative PNU-EDA-Gly₅ (FIG. 3A) was provided byLevana Biopharma, San Diego, Calif., which synthesized a pentaglycinpeptide to the carbonyl group of PNU159682 via an ethylenediamino (EDA)linker according to the synthesis scheme of FIG. 3B. For this,commercially available PNU159682 was first oxidized to obtain acarboxylic acid thereof (1 on FIG. 3B) with NaIO₄ in 60% methanol at RTfor 3 hours. Thereafter, N-hydroxysuccidimide (NHS, 46 mg, 400 μmol) andethyl(dimethylaminopropyl) carbodiimide (EDC, 100 mg, 523 μmol) indichloromethane (DCM) were added to a solution of 1 (51 mg, 81 μmol) in6 mL of DCM. After 30 min, the mixture was washed with water (2×6 mL),dried over Na₂SO₄ and evaporated. The residue was then dissolved in 2 mLof dimethylformamide (DMF) prior to addition of the amine (2 on FIG. 3B,55 mg, 81 μmol, as trifluoroacetate salt), followed by addition ofN,N-diisopropylethylamine (DIEA, 504). The mixture was stirred for 1 hprior to addition of piperidine (404), followed by 20 min of additionalstirring. The mixture was purified by HPLC to give PNU-EDA-Gly₅ (3 onFIG. 3B, 34 mg, 44%) as a red solid; MS m/z 955.2 (M+H).

PNU-EDA-Gly₅ was conjugated to mAbs by incubating LPETG-tagged mAbs [10μM] with PNU-EDA-Gly₅, [200 μM] in the presence of 0.62 μM Sortase A in50 mM Hepes, 150 mM NaCl, 5 mM CaCl₂, pH 7.5 for 3.5 h at 25° C. Thereaction was stopped by passing it through a Protein A HiTrap column (GEHealthcare) equilibrated with 25 mM sodium phosphate pH 7.5, followed bywashing with 5 column volumes (CVs) of buffer. Bound conjugate waseluted with 5 CVs of elution buffer (0.1M succinic acid, pH 2.8) with 1CV fractions collected into tubes containing 25% v/v 1M Tris Base toneutralise the acid. Protein containing fractions were pooled andformulated in 10 mM Sodium Succinate pH 5.0, 100 mg/mL Trehalose, 0.1% %w/v Polysorbate or phosphate20 by G25 column chromatography using NAP 25(GE Healthcare) columns according to the manufacturer's instructions.

The aggregate content of each conjugate was assessed by chromatographyon a TOSOH TSKgel G3000SWXL 7.8 mm×30 cm, 5 μm column run at 0.5 mL/minin 10% IPA, 0.2M Potassium Phosphate, 0.25M Potassium Chloride, pH 6.95.The drug loading was assessed both by Hydrophobic InteractionChromatography (HIC) and Reverse-Phase Chromatography. HIC was performedon a TOSOH Butyl-NPR 4.6 mm×3.5 cm, 2.5 μm column run at 0.8 mL/min witha 12 minute linear gradient between A—1.5M (NH₄)₂SO₄, 25 mM NaPi,pH=6.95±0.05 and B—75% 25 mM NaPi, pH=6.95±0.05, 25% IPA. Reverse phasechromatography was performed on a Polymer Labs PLRP 2.1 mm×5 cm, 5 μmcolumn run at 1 mL/min/80° C. with a 25 minute linear gradient between0.05% TFA/H₂O and 0.04% TFA/CH₃CN. Samples were first reduced byincubation with DTT at pH 8.0 at 37° C. for 15 minutes. BothPNU-EDA-Gly₅-based ADCs were predominantly monomeric and haddrug-to-antibody-ratios close to the theoretical maximum of,respectively, 4. Table 2 summarizes the results of the ADCmanufacturing.

TABLE 2 Summary of PNU-EDA-Gly₅-based ADCs manufactured. HC, heavychain; LC, light chain; % mono, % monomer content; DAR,drug-to-antibody-ratio. mAb target HC tag LC tag % mono DAR BrentuximabCD30 Yes Yes 99.6 4.0 Trastuzumab HER-2 Yes Yes 98.2 3.9

Example 2. In Vitro Cytotoxicity Assay with Sortase A-ConjugatedBrentuximab-PNU-EDA-Gly₅ and Trastuzumab-PNU-EDA-Gly₅ ADCs

Cytotoxicity of Brentuximab-PNU-EDA-Gly₅ was investigated usingKarpas-299, a non-Hodgkin's lymphoma cell line expressing high levels ofCD30, and L428, a Hodgkin's lymphoma cell line expressing low tomoderate levels of CD30 (FIG. 4). As controls, efficacy ofcAc10-PNU-EDA-Gly₅ was compared to that of the commercially availableCD30-specific cAc10-vcPAB-MMAE conjugate Adcetris® (as positive control)and the commercially available HER-2-specific Trastuzumab-DM1 conjugateKadcyla® (as negative control). For this, cells were plated on 96-wellplates in 100 μl RPMI/10% FCS at a density of 10⁴ cells per well andgrown at 37° C. in a humidified incubator at 5% CO₂ atmosphere. Afterone day incubation, 25 μl medium was carefully removed from each welland replaced by 25 μl of 3.5-fold serial dilutions of each ADC in growthmedium, resulting in final ADC concentrations ranging from 20 μg/ml to0.25 ng/ml. Each dilution was done in duplicate. After 4 additionaldays, plates were removed from the incubator and equilibrated to roomtemperature. After approximately 30 minutes, 100 μl CellTiter-Glo®Luminescent Solution (Promega, Cat. No G7570) was added to each welland, after shaking the plates at 450 rpm for 5 min followed by a 10 minincubation without shaking, luminescence was measured on a TecanInfinity F200 with an integration time of 1 second per well.

As expected, the anti-CD30 ADC Adcetris® used as a positive controlpotently killed CD30^(HI) Karpas-299 cells with an EC50 of 8.2 ng/ml(FIG. 4A), while being inefficient at killing CD30^(LO) L428 cells (FIG.4B). In contrast, the anti-HER-2 ADC Kadcyla® used as a negative controldisplayed no specific cell killing and was ineffective on either cellline (FIG. 4). Significantly, Sortase-conjugated ADC cAc10-PNU-EDA-Gly₅potently killed the CD30^(HI) Karpas-299 cells with an EC50 value of 6.9ng/ml (FIG. 4A). cAc10-PNU-EDA-Gly₅ killed the CD30^(LO) L428 cells onlyat higher concentrations, similar to the control ADCs employed,indicating that the efficacy of this ADC is indeed specific and mediatedby CD30 binding (FIG. 4B). Thus, Sortase-mediated conjugation ofPNU-EDA-Gly₅ yielded an ADC with a very high potency, even exceedingthat of the reference ADC Adcetris®.

The potency for tumor cell killing of a SMAC-generatedTrastuzumab-PNU-EDA-Gly₅ ADC was investigated using SKBR3 cells, a humanbreast cancer cell line overexpressing HER-2, and T47D cells, a breastcancer cell line naturally expressing low levels of HER-2, and this wascompared to the commercially available HER-2-specific ADCTrastuzumab-DM1 conjugate Kadcyla® (FIG. 5). For this, cells were platedon 96 well plates in 100 μl DMEM/10% FCS at a density of 10⁴ cells perwell and assays were performed exactly as described above.

As expected, the positive control ADC Kadcyla® potently killedHER-2-overexpressing human SKBR3 breast cancer cells, with an EC50 of23.7 ng/ml (FIG. 5A), while being ineffective at killing HER-2^(LO) T47Dcells (FIG. 5B). Significantly, Trastuzumab-PNU-EDA-Gly₅ generated bySMAC-technology displayed superior cytotoxicity and not only killedHER-2-overexpressing SKBR3 cells, but also HER2^(LO) T47D cells, withEC50 values of, respectively, 4.8 and 11.0 ng/ml (FIG. 5). Thus,Sortase-mediated conjugation of PNU-EDA-Gly₅ to Trastuzumab yields anADC with a very high potency, exceeding that of the commerciallyavailable and FDA-approved reference ADC Kadcyla®, and is even effectiveon HER2^(LO) human breast cancer cells.

Example 3: In Vitro Serum Stability of Sortase A-ConjugatedcAc10-PNU-EDA-Gly₅ ADC as Compared to Maleimide Linker ContainingTrastuzumab Emtansine (Kadcyla®)

The in vitro serum stability of brentuximab-PNU-EDA-Gly₅(cAc10-PNU-EDA-Gly₅) and Kadcyla ADCs was evaluated in an ELISA-basedserum stability assay. Briefly, cAc10-PNU-EDA-Gly₅ was diluted in mouse(Sigma, M5905), rat (Sigma, R9759) and human serum (Sigma, H6914), andincubated at 37° C. Samples were snap-frozen in liquid nitrogen on days0, 3, 7, 14 and stored at −80° C. until ELISA analysis. For rodent sera,dilution series of cAc10-PNU-EDA-Gly₅ serum samples were captured onELISA plates coated with 2 μg/ml of a mouse anti-PNU mAb (producedin-house by immunizing mice with a human IgG-PNU conjugate and screeningwith a BSA-PNU conjugate) to bind ADC, or with anti-human Fc F(ab′)2(Jackson Immunoresearch) to bind total IgG, and detected with a 1:2500dilution of an HRP-conjugated anti-human IgG F(ab′)2 (JacksonImmunoresearch). For primate sera, 2 μg/ml of recombinant human CD30(Sino Biologicals, 10777-H08H) was coated on ELISA plates and a 1:2500dilution of HRP-conjugated anti-human IgG F(ab′)2 (JacksonImmunoresearch) or 1 μg/ml of a mouse anti-PNU IgG (produced in-house)followed by HRP-conjugated anti-mouse Fc F(ab′)2 (JacksonImmunoresearch) was used for detection of total IgG and ADC,respectively. In the case of Kadcyla, the same protocol was used asabove to determine stability in mouse, rat and human serum but with anin-house produced anti-maytansine mAb to bind ADC. Serum concentrationsof ADC and total IgGs were calculated from half maximal values of thesample titrations by comparison with a sample of the same ADC of knownconcentration.

FIG. 7 A shows the excellent stability of cAc10-PNU-EDA-Gly₅ ADC,particularly as compared to that of maleimide linker containing Kadcyla(FIG. 7 B), with virtually no decrease in ADC levels throughout theentire experiment in any serum of the four species tested. By fittingthe time points between day 0 and 14 to a one-phase exponential decayfunction constrained to reach a final concentration of 0, the half-lifevalues of cAc10-PNU-EDA-Gly₅ and Kadcyla were determined in each serum.The half-life of Kadcyla was of 3.7 days, 4.4 days and 2.9 days inmouse, rat and human serum, respectively, whereas the half-life ofcAc10-PNU-EDA-Gly₅ was greater than 14 days in mouse, rat and humanserum.

Example 4: In Vivo Stability of Sortase A-Conjugated Ac10-Gly5-PNU inMice

Ac10-Gly5-PNU ADC was thawed at room temperature and diluted to 0.2mg/ml in sterile PBS for a dosing concentration of 1 mg/kg. The sampleswere injected i.V. at a volume of 5 mL/kg in nine female Swiss Webstermice. Blood was collected from animals after 1 h, 24 h, 72 h, 7 days, 14days, and 21 days. Individual animals according to ethical standardswere only used for two blood draw time points at least a week apart.Thus, three mice had blood drawn after 1 h and 7 days, three differentmice had blood drawn after 24 h and 14 days, and three additionaldifferent mice had blood drawn after 72 h and 21 days for a total ofnine mice per group. For each group of animals, approximately 2004, ofblood was collected by lancet-puncture of the submandibular vein duringthe first collection, and approximately 6004, of blood bylancet-puncture of submandibular vein during the final collection(terminal bleed). All blood was collected into tubes containing K2-EDTA.Plasma was isolated from blood by centrifugation at 1500 g for 10minutes, and transferred to sterile cryovials for storage at −80° C.until analysis by ELISA as described in Example 4.

The data in FIG. 8 shows the high stability of the ADC generated bySMAC-technology. For the entire duration of the experiment,concentrations of ADC are only marginally lower than those measured fortotal IgG, which implies that the linker between drug and antibody isstable in vivo. By fitting the time points between day 3 and 21 to aone-phase exponential decay function constrained to reach a finalconcentration of 0, in vivo half life in the slow phase was determinedwith 8.3 and 7.8 days for total IgG and ADC, respectively.

Example 5: Description and Characterization of EMT-6 Clones ExpressingHER-2

Cytotoxicity of anti-HER-2 ADCs was investigated using the murinemammary tumor cell line EMT-6 engineered to overexpress human HER-2.EMT-6 cells were cultured as monolayers in DMEM (Dulbecco's ModifiedEagle Medium—high glucose) supplemented with 10% (v/v) of FCS (FetalCalf Serum), 1% (v/v) of 10,000 IU/mL penicillin-streptomycin and 1%(v/v) of 200 mM L-glutamine.

EMT-6 cells were electroporated with an expression vector encoding thehuman HER-2 gene and a puromycin resistance marker and cell pools stablyexpressing human HER-2 were selected using methods known to thoseskilled in the art.

HER-2 expression was confirmed by flow cytometry. Briefly, followingtrypzinization, 10⁶ cells were centrifuged in FACS tubes; obtainedpellets were resuspended in PBS (phosphate-buffered saline) supplementedwith 2% of FCS. Cells were then incubated with the anti-HER-2 antibodytrastuzumab (30 min, 4° C.), followed by centrifugation and washing (3mL of PBS with 2% FCS). Cells were then resuspended as previously andincubated with anti-human IgG antibody (F_(c) gamma-specific) PE(Ebioscience) in the dark (30 min, 4° C.), prior to washing (4 mL PBSwith 2% FCS). Flow cytometry was then performed on a FACS Calibur (BD).

HER-2-transfected EMT-6 cells were single cell-sorted by flow cytometryusing a FACS ARIA II to isolate single cell clones. These were expandedand HER-2 expression was verified by flow cytometry.

FIG. 9 shows the FACS analysis data of the clone selected for in vivostudies (Example 6).

Example 6: In Vivo Efficacy of Sortase A-ConjugatedTrastuzumab-PNU-EDA-Gly₅ ADC in an Orthotopic Breast Cancer Model

The in vivo efficacy of Trastuzumab-PNU-EDA-Gly₅ was evaluated in animmunocompetent orthotopic mouse model of HER-2-positive breast cancer.For this, 10⁶ EMT6 mouse breast cancer cells expressing human HER-2(Example 6), previously determined to be suitable for in vivo growth,were implanted into the right mammary fat pads of female Balb/c mice. Inaddition, control animals were implanted with HER-2-negative EMT6 cells.In the following, primary tumor volumes were measured by calipering.After 13 days, when a mean tumor volume of 100-150 mm³ was reached,tumor-bearing animals were randomized into groups of 6 animals eachaccording to tumor sizes. Animals were treated on the same day (day 13,i.e. day of randomization) and 7 days later (day 20) by intravenousinjection of the reference ADC Kadcyla® (15 mg/kg),Trastuzumab-PNU-EDA-Gly₅ (1 mg/kg) or vehicle control. Tumor sizes weremonitored by calipering and animals whose tumor volume reached 1000-1500mm³ were terminated (FIG. 10).

Tumors in vehicle control mice grew rapidly and reached an average sizeof approximately 1000 mm³ within 30 days after transplantation of cells(FIG. 10A). Treatment with Kadcyla® had little effect on tumor growth inmost animals. Only one out of six animals displayed a significant delayin tumor growth (FIG. 10C). In striking contrast, in all animals treatedwith Trastuzumab-PNU-EDA-Gly₅, the tumors continuously regressed duringtreatment and were essentially undetectable by day 30 aftertransplantation of the cells (FIG. 10D). No tumor was detectable mostanimals until day 60, and tumor recurrence was observed in only oneanimal around day 40. Significantly, the anti-tumor activity ofTrastuzumab-PNU-EDA-Gly₅ was highly specific and treatment of micebearing HER-2-negative tumors did not lead to tumor regression (FIG.10B). Taken together, the data demonstrate that sortase-mediatedsite-specifically conjugated of Trastuzumab-EDA-Gly₅-PNU ADCs yielded anADC with in vivo tumor cell killing activity far superior to thebenchmark ADC Kadcyla®.

FIGURE LEGENDS

FIG. 1: Schematic drawing of site-specific sortase mediated antibodyconjugation (SMAC-technology). The monoclonal antibodies need to beproduced with C-terminal LPXTG sortase tags. The toxic payload needs tobe produced to contain an oligoglycine peptide stretch (Gly_(n)-stretch)with a certain number of glycine residues in a row (n≧1 and ≦21,preferably n≧3 and ≦10, preferably n=3 or n=5, most preferably n=5).Sortase A enzyme from Staphylococcus aureus specifically recognizes theLPXTG pentapeptide motif and catalyzes the transpeptidation of theoligo-glycine peptide stretch to the threonine-glycine peptide bond ofLPXTG, thereby generating a new stabile peptide bond between thethreonine and the N-terminal glycine of the oligo-glycine stretch.

FIG. 2. Structure of PNU-159682 as described in the prior art (e.g.WO2009099741, or Quintieri et al (2005)), including the officialanthracycline numbering system for reactive carbons of the tetracyclicaglycone structure.

FIG. 3. (A) Structure of PNU derivative-EDA-Gly₅, called “PNU-EDA-Gly₅”herein, as utilized for the SMAC-technology conjugation to C-terminallyLPETG sortase tagged monoclonal antibodies using sortase enzyme asdisclosed in the Examples herein. (B) Synthesis scheme of anthracyclinederivative PNU-EDA-Gly₅.

FIG. 4. Dose response of the cytotoxic effects of the indicated ADCs onhuman Non-Hodgkin lymphoma cell line Karpas-299, expressing high levelsof CD30 target on the cell surface (A), and on human Hodgkin lymphomacell line L428 cells expressing very low levels of CD30 target in thecell surface (B). Adcetris refers to commercially available anti-CD30ADC brentuximab-vedotin. Kadcyla refers to commercially availableanti-HER-2/neu ADC T-DM1 (trastuzumab-emtansine). Both cell lines arenegative for HER-2/neu, and therefore Kadcyla acts as a negative controlADC, that should not effect cell killing in a target-specific way. Cellswere incubated with serial dilutions of ADCs for 4 days, after whichCellTiter-Glo® Luminescent Solution (Promega) was added and viable cellswere quantified by measuring the luminescence on a Tecan Infinity F200.

FIG. 5. Dose response of the cytotoxic effects of the indicated ADCs onhuman breast cancer cell line SKBR3, expressing high levels of HER-2/neu(A) and human breast cancer cell line T47D expressing low levels ofHER-2/neu (B). Cells were incubated with serial dilutions of ADCs for 4days, after which CellTiter-Glo® Luminescent Solution (Promega) wasadded and viable cells were quantified by measuring the luminescence ona Tecan Infinity F200.

FIG. 6. Additional PNU-159682 related anthracycline derivatives usefulfor site-specific-conjugation to LPXTG-tagged binding proteins orantibodies by SMAC-technology to produce BPDCs or ADCs. Only thepreferred versions with Gly5-stretch are depicted. 6A depicts aderivative, in which the Gly5 amino acid stretch is directly coupled viaits carboxy terminus to the A-Ring of the tetracyclic aglycone structureof the PNU derivative. 6B depicts a derivative in which a preferredethylene-amino linker and Gly5 amino acid stretch is directly coupled tothe A-Ring of the tetracyclic aglycone structure of the PNU derivative

FIG. 7 (A) Measurement of in vitro concentration ofbrentuximab-PNU-EDA-Gly₅ ADC (labeled as “cAc10-PNU ADC”) and total IgGin mouse (A), rat (B), human (C) serum over 14 days. (B) Measurement ofin vitro concentration of trastuzumab-emtansine (Kadcyla®) ADC and totalIgG in mouse (A), rat (B) and human (C) serum over 14 days.

FIG. 8: In vivo plasma concentrations of ADC and total IgG measured at 6time-points over a 21-day period following administration ofAc10-Gly5-PNU ADC in mice.

FIG. 9: Data of FACS analysis of EMT-6 HER-2 clone selected for in vivostudies following incubation with anti-HER-2 antibody trastuzumab andthen incubation with flurophore-containing anti-human IgG antibody (Fcgamma-specific) PE.

FIG. 10: In vivo evaluation of HER-2-specific ADCs in an immunocompetentorthotopic mouse model of HER2-positive breast cancer. EMT6 mouse breastcancer cells expressing human HER-2 (A, C, D) or irrelevant antigenROR-1 were grown in the mammary fat pads of Balb/c mice. On days 13 and20, animals were treated i.v. with vehicle control (A), 1 mg/kgTrastuzumab-PNU159682 (B, D), or 15 mg/kg Kadcyla (C). Tumor growth wasmonitored until animals had to be sacrificed due to ethical reasons.

FIGS. 11 A & B: Amino acid compositions of the C-terminallySMAC-Technology™ conjugated IgH and IgL chains of the trastuzumab (A)and brentuximab (B) PNU-toxin derivative containing ADCs used for thestudies, comprising the PNU derivative depicted in FIG. 3B linkedthrough the amino group of the Gly5-stretch to the 4th amino acid of thesortase tag (highlighted in boldface print) via a peptide bond followingsortase enzyme conjugation.

REFERENCES

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1. An anthracycline (PNU) derivative conjugate or a binding protein-drugconjugate (BPDC) the same, said conjugate comprising a derivative of theanthracycline PNU-159682 having the following formula (i) or formula(ii)

said conjugate comprising at its wavy line a linker structureX-L₁-L₂-L₃-Y, wherein L₁-L₃ represent linkers, and two of L₁-L₃ aremandatory, and wherein X and Y further represent each one or moreoptional linkers.
 2. A binding protein-drug conjugate (BPDC), having thefollowing formula:

wherein a) L₁-L₃ represent linkers, and two of L₁-L₃ are mandatory, b) Xany Y each represent one or more optional linkers, c) BP is a bindingprotein, and d) n is an integer ≧1 and ≦10.
 3. The anthracycline (PNU)derivative conjugate according to claim 1, wherein the linker structurecomprises, as L₂, an oligo-glycine peptide (Gly)_(n) coupled to saidanthracycline derivative, directly or by means of another linker L₁, insuch a way that the oligo-glycine (Gly)_(n) peptide has a free aminoterminus, and wherein n is an integer ≧1 and ≦21.
 4. The anthracycline(PNU) derivative conjugate or the binding protein-drug conjugate (BPDC)according to claim 1, wherein the oligo-glycine peptide (Gly)_(n) isconjugated to the anthracycline derivative of formula (i) by means of analkylenediamino linker (EDA), designated as L₁, which alkylenediaminolinker is conjugated to the anthracycline derivative by means of a firstamide bond, while it is conjugated to the carboxy terminus of theoligo-glycine peptide by means of a second amide bond, said conjugate ofalkylenediamino linker and oligo-glycine peptide having the followingformula (v),

wherein the wavy line indicates the linkage to the anthracyclinederivative of formula (i), wherein m is an integer ≧1 and ≦11 and n isan integer ≧1 and ≦21.
 5. The anthracycline (PNU) derivative conjugateor the binding protein-drug conjugate (BPDC) according to claim 3,wherein the oligo-glycine peptide (Gly)_(n) is, directly or by means ofanother linker L₁, coupled to Ring A of the anthracycline derivative offormula (ii).
 6. The anthracycline (PNU) derivative conjugate or thebinding protein-drug conjugate (BPDC) according to claim 3, wherein theoligo-glycine peptide (Gly_(n)) is conjugated to the anthracyclinederivative of formula (ii) by means of an alkyleneamino linker (EA),designated as L₁, which alkyleneamino linker is conjugated to thecarboxy terminus of the oligo-glycine peptide by means of an amide bond,said conjugate of alkyleneamino linker and oligo-glycine peptide havingthe following formula (vi)

wherein the wavy line indicates the linkage to the anthracyclinederivative of formula (ii), wherein m is an integer ≧1 and ≦11 and n isan integer ≧1 and ≦21.
 7. The binding protein-drug conjugate (BPDC)according to claim 3, wherein the linker structure L₃ comprises apeptide motif that results from specific cleavage of a sortase enzymerecognition motif.
 8. The binding protein-drug conjugate (BPDC)according to claim 7, wherein said sortase enzyme recognition motifcomprises a pentapeptide.
 9. The binding protein-drug conjugate (BPDC)according to claim 7, wherein said sortase enzyme recognition motifcomprises at least one of the following amino acid sequences LPXTG,LPXSG, and/or LAXTG.
 10. The binding protein-drug conjugate (BPDC)according to claim 2, wherein the anthracycline (PNU) derivative isconjugated, by means of the one or more linkers, to the carboxy terminusof the binding protein, or to the carboxy terminus of at least onedomain or subunit thereof.
 11. The binding protein-drug conjugate (BPDC)according to claim 2, wherein the binding protein is conjugated to thefree amino terminus of the oligo-glycine peptide (Gly_(n)) by means ofan amide bond.
 12. The binding protein-drug conjugate (BPDC) accordingto claim 2, wherein the binding protein is at least one selected fromthe group consisting of an antibody, modified antibody format, antibodyderivative or fragment, antibody-based binding protein, oligopeptidebinder and an antibody mimetic.
 13. The binding protein-drug conjugate(BPDC) according to claim 2, wherein the binding protein binds at leastone entity selected from the group consisting of a receptor, an antigen,a growth factor, a cytokine, and/or a hormone.
 14. The bindingprotein-drug conjugate (BPDC) according to claim 2, wherein the bindingprotein has at least two subunits.
 15. The binding protein-drugconjugate (BPDC) according to claim 14, wherein at least one subunitcomprises a derivative of the anthracycline PNU-159682.
 16. The bindingprotein-drug conjugate (BPDC) according to claim 2, wherein the bindingprotein binds HER-2.
 17. The binding protein-drug conjugate (BPDC)according to claim 16, wherein the binding protein is an antibody thatbinds HER-2.
 18. The binding protein-drug conjugate (BPDC) according toclaim 2, wherein the antibody is characterized as follows: a) comprisesthe CDR regions 1-6 of trastuzumab; b) comprises the heavy chainvariable domain and the light chain variable domain of trastuzumab; c)has an amino acid sequence identity of 90% or higher with the regions ordomains of a) or b); d) is trastuzumab, or a target binding fragment orderivative thereof, and/or e) competes with trastuzumab for binding toHER-2.
 19. The binding protein-drug conjugate (BPDC) according to claim2, wherein the binding protein binds CD30.
 20. The binding protein-drugconjugate (BPDC) according to claim 19, wherein the binding protein isan antibody that binds CD30.
 21. The binding protein-drug conjugate(BPDC) according to claim 19, wherein the antibody is characterized asfollows: a) comprises the CDR regions 1-6 of brentuximab; b) comprisesthe heavy chain variable domain and the light chain variable domain ofbrentuximab; c) has an amino acid sequence identity of 90% or higherwith the regions or domains of a) or b); d) is brentuximab or a targetbinding fragment or derivative thereof, and/or e) competes withbrentuximab for binding to CD30.
 22. A method of producing a bindingprotein-drug conjugate (BPDC) according to claim 2, wherein a bindingprotein carrying a sortase enzyme recognition motif is conjugated, bymeans of a sortase enzyme, to at least one anthracycline derivativeconjugate according to claim 2, which carries, as L₂, an oligo-glycinepeptide (Gly)_(n).
 23. Use of a binding protein drug conjugate (BPDC)according to claim 2, for the treatment of a human or animal subjectsuffering from, at risk of developing, and/or being diagnosed for agiven pathologic condition.
 24. Use according to claim 23, wherein thepathologic condition is a neoplastic disease.
 25. Use according to claim24, wherein the neoplastic disease is a) a cancer that has an HER-2expression score of 1+, 2+ or 3+, as determined by IHC or ISH, whichcancer is preferably a breast cancer, or b) a cancer that is CD30positive as determined by IHC, ELISA or flow cytometry, preferably alymphoma, more preferably a Hodgkin lymphoma (HL) or a systemicanaplastic large cell lymphoma (sALCL).
 26. A pharmaceutical compositioncomprising a binding protein drug conjugate (BPDC) according to claim 2and at least one other pharmaceutically acceptable ingredient.